1. Filed of the Invention
The present invention relates to an indirect agglutination immunoassay and an apparatus therefor, and more particularly to an indirect agglutination immunoassay using an antigen-antibody reaction and magnetic particles or magnetic-material-containing particles, and an apparatus for conducting the indirect agglutination immunoassay.
2. Discussion of Background
Indirect agglutination used in an immunoassay, in which the combining reaction by the antigen-antibody reaction is intensified by use of antigen- or antibody-bonded particles, is called "passive agglutination" or "reverse passive agglutination" and is widely used in practice in a simple immunoassay for a large number of test samples.
The immunoassay utilizing indirect agglutination has the advantages over the conventional EIA (enzyme immunoassay) and RIA (radioimmunoassay) in that the assay is simple in operation and does not require any particular device for detecting the occurrence of the antigen-antibody reaction. However the immunoassay utilizing indirect agglutination has the drawbacks that it is extremely difficult to conduct the assay automatically, and it has not as yet been developed beyond the semi-automation stage.
There are two methods for forming detection patterns during indirect agglutination.
In one method, a particle-containing reagent is added to a diluted solution of a test sample containing a desired analyte placed in a "U" well or "V" well microplate, the mixture is stirred and then allowed to stand, and the occurrence of an antigen-antibody reaction is detected from a sedimentation pattern of the particles of the reagent formed at the bottom of the well. Hereinafter this method is referred to as "the standing method".
In another method, a particle-containing reagent is added to a diluted solution of the test sample placed in a "U" well or "V" well microplate, and the mixture is stirred and then centrifuged to precipitate the particles onto the bottom of the well. The microplate is then inclined, so that the presence or absence of an antigen-antibody reaction is detected from the absence or presence of slippage observed of a coating of the precipitated particles at the bottom of the well of the microplate. Hereinafter this method is referred to as "the centrifugation method".
In the case where the occurrence of an antigen-antibody reaction is detected from the sedimentation pattern by the standing method, it is extremely difficult to detect the pattern automatically because the pattern is easily distorted by slight vibrations during the standing thereof. As a result, for instance, the pattern is deformed, the area of the pattern is decreased, and slippage of the pattern along the bottom of the well takes place. In addition, the standing method has the drawback that about 0.5 to 3 hours are required before the pattern is formed in a suitable fashion for the assay, although the necessary time period for this of course depends upon the type of particles employed.
In contrast, in the case of the centrifugation method, the sedimentation can be finished within a few minutes by use of a centrifuge, and the pattern can be read after the microplate is slanted for several minutes. Furthermore, the concern about the distortion of the pattern caused by vibrations applied thereto during the formation of the pattern is entirely unnecessary. However, it is difficult to perform the immunoassay automatically by use of a centrifuge in practice.
As test samples that can be used for the above-mentioned conventional standing method and centrifugation method, for instance, blood serum, urine, and other body fluids can be given. In conventional methods, the test samples are usually diluted and used. However, when whole blood is used without separating out of the blood corpuscles and blood serum, the sedimentation pattern tends to be centered at one point of the bottom of the well of the microplate, so that the blood components adversely affect the shape of the agglutination or sedimentation pattern. It is known that this will distort the results of the assay.
In other conventional immunoassays, such as the EIA (enzyme immunoassay) and RIA (radioimmunoassay), the separation of blood corpuscles and blood serum is conducted as a pre-processing step in order to avoid non-specific reactions.